Masamichi Nakayama

Controlled Chain Length and Graft Density of Thermoresponsive Polymer Brushes for Optimizing Cell Sheet Harvest

Novel thermoresponsive polymer brush surfaces for harvesting cell sheet were fabricated by the surface-initiated RAFT polymerization of N-isopropylacrylamide (IPAAm) on azoinitiator-immobilized glass substrates in the presence of dithiobenzoate compound as a chain transfer agent (CTA). The chain length of the grafted PIPAAm on the surface was controlled by changing CTA concentration. Additionally, PIPAAm graft density on the surface was successfully regulated by grafting from azoinitiator-immobilized surfaces with various densities. By adjusting both the chain length and the density of grafted PIPAAm, a series of thermoresponsive polymer brush surfaces were prepared to regulate cell adhesion/detachment behavior by solely temperature change across the PIPAAms lower critical solution temperature of 32 degrees C. PIPAAm brush surfaces were successfully optimized to recover the cell sheets of bovine carotid artery endothelial cells. Additionally, the immunostaining study revealed that the cell sheets can be recovered with their intact extracellular matrix (ECM) from PIPAAm surfaces, indicating that the cell sheets can be effectively transplanted to damaged tissues and organs.

Temperature-Induced Intracellular Uptake of Thermoresponsive Polymeric Micelles

Well-defined diblock copolymers comprising thermoresponsive segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(IPAAm-co-DMAAm)) and hydrophobic segments of poly(d,l-lactide) were synthesized by combination of RAFT and ring-opening polymerization methods. Terminal conversion of thermoresponsive segments was achieved through reactions of maleimide or its Oregon Green 488 (OG) derivative with thiol groups exposed by cleavage of polymer terminal dithiobenzoate groups. Thermoresponsive micelles obtained from these polymers were approximately 25 nm when below the lower critical solution temperature (LCST) of 40 degrees C, and their sizes increased to an average of approximately 600 nm above the LCST due to aggregation of the micelles. Interestingly, the OG-labeled thermoresponsive micelles showed thermally regulated internalization to cultured endothelial cells, unlike linear thermoresponsive P(IPAAm-co-DMAAm) chains. While intracellular uptake of P(IPAAm-co-DMAAm) was extremely low at temperatures both below and above the micellar LCST, the thermoresponsive micelles showed time-dependent intracellular uptake above the LCST without exhibiting cytotoxicity. These results indicate that the new thermoresponsive micelle system may be a greatly promising intracellular drug delivery tool.

Temperature-responsive polymeric micelles for optimizing drug targeting to solid tumors

Targeting to solid tumors is the most challenging issue in the drug delivery field. To obtain the ideal pharmacodynamics of administrated drugs, drug carriers must suppress drug release and interactions with non-target tissues while circulating in the bloodstream, yet actively release the incorporated drug and interact with target cells after delivery to the tumor tissue. To handle this situation, stimuli-responsive drug carriers are extremely useful, because carriers change their physicochemical properties to control the drug release rate and interaction with cells in response to the surrounding environmental conditions or applied physical signals. The current review focuses on the strategy and availability of temperature-responsive (TR) polymeric micelles as a next-generation drug carrier. In particular, we discuss the unique properties of TR polymeric micelles, such as temperature-triggered drug release and intracellular uptake system. In addition, we explore the methodology for integrating other targeting systems into TR micelles to pursue the ideal pharmacodynamics in conjunction with thermal therapy as a future prospective of the TR system.

Terminally Functionalized Thermoresponsive Polymer Brushes for Simultaneously Promoting Cell Adhesion and Cell Sheet Harvest

For preparing cell sheets effectively for cell sheet-based regenerative medicine, cell-adhesion strength to thermoresponsive cell culture surfaces need to be controlled precisely. To design new thermoresponsive surfaces via a terminal modification method, thermoresponsive polymer brush surfaces were fabricated through the surface-initiated reversible addition-fragmentation chain transfer (RAFT) radical polymerization of N-isopropylacrylamide (IPAAm) on glass substrates. The RAFT-mediated grafting method gave dithiobenzoate (DTB) groups to grafted PIPAAm termini, which can be converted to various functional groups. In this study, the terminal carboxylation of PIPAAm chains provided high cell adhesive property to thermoresponsive surfaces. Although cell adhesion is generally promoted by a decrease in the grafted PIPAAm amount, the decrease also decelerated thermally-induced cell detachment, whereas the influence of terminal modification was negligible on the cell detachment. Consequently, the terminally modified PIPAAm brush surfaces allowed smooth muscle cells (SMCs) to simultaneously adhere strongly and detach themselves rapidly. In this study, SMCs were unable to reach a confluent monolayer on as-prepared PIPAAm brush surfaces (grafted amount: 0.41 μg/cm(2)) without terminal carboxylation due to their insufficient cell-adhesion strength. On the other hand, though a decrease in the PIPAAm amount allowed SMCs to form a confluent cell monolayer on the PIPAAm brush surface, the SMCs were unable to be harvested as a monolithic cell sheet by low-temperature culture at 20 °C. Because of their unique property, only terminal-carboxylated PIPAAm brush surfaces achieved rapid harvesting of complete cell sheets by low-temperature culturing.

The use of anisotropic cell sheets to control orientation during the self-organization of 3D muscle tissue ☆

In some parts of native tissues, the orientation of cells and/or extracellular matrixes is well organized. We know that because anisotropy produces important tissue functions, an appropriate anisotropy needs to be designed to biomimetically construct complex tissue. Here, we show the unique features of anisotropic myoblast sheets for organizing the three-dimensional (3D) orientation of myoblasts and myotubes. Utilizing a micropatterned thermoresponsive surface, human skeletal muscle myoblasts were aligned on the surface, and manipulated as a single anisotropic cell sheet by reducing the culture temperature. Consequently, layering of anisotropic myoblast sheets using gelatin gel allowed 3D myotube constructs to be built up with the desired anisotropy. We also discovered a surprising myoblast behavior. An anisotropic cell sheet placed on top of other cell sheets in fabricating thick tissue was able to change the cell orientation in several layered cell sheets underneath. This self-organization is believed to provide the uniqueness required in designing 3D cell orientation architecture for reconstructed muscle tissue.

Anisotropic cell sheets for constructing three-dimensional tissue with well-organized cell orientation

Normal human dermal fibroblasts were aligned on micropatterned thermoresponsive surfaces simply by one-pot cell seeding. After they proliferated with maintaining their orientation, anisotropic cell sheets were harvested by reducing temperature to 20 °C. Surprisingly, the cell sheets showed different shrinking rates between vertical and parallel sides of the cell alignment (aspect ratio: approx. 3: 1), because actin fibers in the cell sheets were oriented with the same direction. The control of cell alignment provided not only a physical anisotropy but also biological impacts to the cell sheet. Vascular endothelial growth factor (VEGF) secreted by aligned fibroblasts was increased significantly, whereas transforming growth factor-β1 (TGF-β1) expression was the same level in anisotropic cell sheets as cell sheets having random cell orientations. Furthermore, although the amount of deposited type Ⅰ collagen was different non-significantly onto between cell sheets with and without controlled cell alignment, collagen deposited onto fibroblasts sheets with cell alignment also showed anisotropy, verified by a fluorescence imaging analysis. The physical and biological anisotropies of cell sheets were potentially useful to construct biomimetic tissues that were organized by aligned cells and/or extracellular matrix (ECM) including collagen in cell sheet-based regenerative medicine. Furthermore, due to the unique thermoresponsive property, the anisotropic cell sheets were successfully manipulated using a gelatin-coated plunger and were layered with maintaining their cell alignment. The combined use of the anisotropic cell sheet and cell sheet manipulation technique promises to create complex tissue that requires the three-dimensional control of their anisotropies, as one of the next-generation cell sheet technologies.

Thermally Controlled Intracellular Uptake System of Polymeric Micelles Possessing Poly(N-isopropylacrylamide)-Based Outer Coronas

Temperature-induced intracellular uptake mechanism of thermoresponsive polymeric micelles comprising poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(d,l-lactide) (P(IPAAm-DMAAm)-b-PLA) inside cultured bovine carotid endothelial cells is investigated by flow cytometry and confocal laser scanning microscopy. Hydrodynamic sizes of P(IPAAm-DMAAm)-b-PLA micelles are approximately 20 nm below the lower critical solution temperature (LCST) of 39.4 degrees C, and their sizes increased to ca. 600 nm above the LCST due to the aggregation of micelles. Intracellular uptake of P(IPAAm-DMAAm)-b-PLA micelles is significantly limited at a temperature below the micellar LCST, 37 degrees C. Of great interest, the P(IPAAm-DMAAm)-b-PLA micelles are internalized into the cells above the micellar LCST (42 degrees C), being dependent on polymer concentration, time, and temperature. By contrast, no intracellular uptake of polyethylene glycol-b-PLA micelles is observed regardless of temperature changes. Enhanced intracellular micelle uptake is probably due to the enhanced interactions between the micelles and cell membranes through the dehydration of corona-forming thermoresponsive polymer chains. Internalization of submicrometer-scale micellar aggregates inside the cells is probably due to their various endocytosis mechanisms. P(IPAAm-DMAAm)-b-PLA micelles localize at the Golgi apparatus and endoplasmic reticulum, but not inside lysosomes. These results indicate that the thermoresponsive polymeric micelles are greatly promising as intracellular delivery tools of drugs, nucleic acids, and peptides/protein without lysosomal decomposition in conjunction with applied heating.

Thermoresponsive poly(N-isopropylacrylamide)-based block copolymer coating for optimizing cell sheet fabrication.

Thermoresponsive surfaces are prepared via a spin-coating method with a block copolymer consisting of poly(N-isopropylacrylamide) (PIPAAm) and poly(butyl methacrylate) (PBMA) on polystyrene surfaces. The PBMA block suppresses the removal of deposited PIPAAm-based polymers from the surface. The polymer coating affects the temperature-dependent cellular behavior of the surfaces with respect to protein adsorption. By adjusting layer thicknesses, PBMA-b-PIPAAm-coated surfaces are optimized to regulate the adhesion/detachment of cells by temperature changes. Thus, thermoresponsive polymer-coated surfaces are able to harvest contiguous cell sheets with their basal extracellular matrix proteins.

Polymeric micelles with stimuli-triggering systems for advanced cancer drug targeting

Abstract Since the 1990s, nanoscale drug carriers have played a pivotal role in cancer chemotherapy, acting through passive drug delivery mechanisms and subsequent pharmaceutical action at tumor tissues with reduction of adverse effects. Polymeric micelles, as supramolecular assemblies of amphiphilic polymers, have been considerably developed as promising drug carrier candidates, and a number of clinical studies of anticancer drug-loaded polymeric micelle carriers for cancer chemotherapy applications are now in progress. However, these systems still face several issues; at present, the simultaneous control of target-selective delivery and release of incorporated drugs remains difficult. To resolve these points, the introduction of stimuli-responsive mechanisms to drug carrier systems is believed to be a promising approach to provide better solutions for future tumor drug targeting strategies. As possible trigger signals, biological acidic pH, light, heating/cooling and ultrasound actively play significant roles in signal-triggering drug release and carrier interaction with target cells. This review article summarizes several molecular designs for stimuli-responsive polymeric micelles in response to variation of pH, light and temperature and discusses their potentials as next-generation tumor drug targeting systems.

Terminal-functionality effect of poly(N-isopropylacrylamide) brush surfaces on temperature-controlled cell adhesion/detachment.

Terminally functionalized poly(N-isopropylacrylamide) (PIPAAm) brush grafted glass surfaces were prepared by a surface-initiated reversible addition-fragmentation chain transfer radical (SI-RAFT) polymerization. SI-RAFT mediated PIPAAm chains possessed terminal dodecyl trithiocarbonate groups which can be substituted with various functional groups. In this study, dodecyl groups were substituted with hydrophilic maleimide groups for controlling the thermoresponsive character of PIPAAm brushes. PIPAAm brushes exhibited reversible temperature-dependent surface wettability changes around PIPAAms lower critical solution temperature. Phase transition of dodecyl-terminated PIPAAm brushes clearly shifted to lower temperature than that of maleimide-terminated PIPAAm brushes, and this shift was attributed to promoted PIPAAm dehydration via terminal hydrophobes. By using this feature, the specific adhesion temperatures of bovine carotid artery endothelial cells (BAECs) on the PIPAAm brush surfaces were successfully controlled. BAECs were initiated to adhere on dodecyl-PIPAAm surfaces at 31 °C, while their adhesion was significantly suppressed on maleimide-PIPAAm surfaces under 33 °C. In contrast, terminal functionality scarcely affected the thermoresponsive behavior of PIPAAm brushes in the polymer rehydration process by reducing temperatures, and thus, the difference in spontaneous cell detachment from different PIPAAm-brush surface was negligible. Consequently, confluently cultured cells were able to be harvested as contiguous cell sheets from individual surfaces with comparable periods at 20 °C.